A recently identified family of intracellular proteins termed Smads, are key components of TGFbeta signaling pathways. Phosphorylation of Smad proteins by activated TGFbeta-type I receptors lead to their translocation to the nucleus where they act as transcriptional regulators. Different Smad proteins act as downstream effectors in response to specific TGFbeta-related ligands. Thus Smad1 mediates the effects of Bone Morphogenetic Proteins, whereas Smad2 is activated in response to TGFbeta and activin. This project aims to gain further insight into the functional activities provided by Smad1 and Smad2 proteins during early mammalian development. A null mutation in the mouse Smad2 gene results in early embryonic lethality. I propose to characterize the primary tissue defects in Smad2 deficient embryos. I plan to isolate Smad2 deficient cell lines. Their abilities to respond to a variety of TGFbeta molecules will be analyzed. To evaluate the requirement of the Smad2 pathway in mesoderm formation, the differentiation abilities of Smad2 deficient ES cell lines will be tested in vitro and in chimeras. Additionally I plan to create Smad1 deficient embryos using gene targeting techniques. Their phenotype will be compared with that of the Smad2 mutants. Overall, results of these experiments will further our understanding of the diverse roles played by distinct Smad pathways during establishment of the embryonic body plan.